Original study - ZZI 01/2011

Comparative microarray analyses of inflamed human periimplant and periodontal tissues in vivo

The ECM undergoes constant physiological remodeling and degradation, which is determined by proteolytic enzymes. However, these enzymes can also be activated in pathophysio-logical processes. Collagenases (including MMPs) play an important part in this [22]. Both biopsies and cultured tissue samples from inflamed gingiva showed increased collagenase activity compared with biopsies from healthy gingiva. In addition, it was shown that an increase in the collagenase activity in the crevicular fluid of patients with gingivitis or periodontitis correlates with progression of this disease [25]. On the basis of this information, attempts were made in different studies to find individual MMPs that stand out from the group of MMPs [16]. Apart from pathogenicity, attention was directed toward the definite occurrence of different MMPs in periodontitis or periimplantitis. In the course of these investigations, Sorsa et al. showed that MMP-8 plays a central role in periodontitis [22].

Apart from the MMPs, cathepsins B and C also play an important part in the inflammatory reaction [27]. The involvement of cathepsins in the destruction of periodontal structures was demonstrated by showing that cathepsins B, D, G and L are increased in chronic periodontitis [5]. An increase in cathepsin K was found in the crevicular fluid in periimplantitis but cathepsin K was not identified as a marker [23]. The pathogenesis of periimplantitis is a possible reason for this. Controlled animal studies showed that bone resorption during progressive periimplantitis is not a linear process [29]. As a result, molecular biological parameters (e. g. cathepsin K) can reflect both the active phase of tissue disintegration and also the resting phase of the disease [23].

Apart from enzyme activity, the breakdown of the ECM is also regulated by the activity and number of inhibitors, which include the group of the TIMPs (tissue inhibitors of metalloproteinases). A series of articles deals with the precise function and regulation of the TIMPs in periodontitis. The studies arrive at different hypotheses as to how the different gene expressions of the TIMPs should be interpreted. Some studies show that a disturbed ratio between MMPs and TIMPs (in favor of the MMPs) can lead to destruction of the periodontal tissue [26]. However, some studies also found an increase in TIMP expression in the inflamed periodontal tissue, which was explained by attempted tissue homeostasis [1]. However, the increase in TIMP expression in periodontitis does not appear adequate to inhibit all up-regulated MMPs sufficiently [8].

Objective

Because of the high proportion of extracellular matrix (ECM) both in the periodontium and in the periimplant bone, the objective of this study was to compare the gene expression of individual ECM proteins and of the enzymes that degrade them using microarrays and real-time PCR in order to identify common features and differences between periodontitis and periimplantitis.

Materials and methods

Patients

The tissue samples were derived from 48 patients (21 men, 27 women, aged between 18 and 72 years), who were undergoing dental treatment in Göttingen University Hospital. A detailed medical history was obtained from each patient. Patients who had systemic diseases such as coronary heart disease, diabetes mellitus or syndromes were excluded from the study. Nicotine consumption was a further exclusion criterion. The tissue samples for the healthy mRNA pool were taken from healthy teeth (n = 16) extracted during orthodontic treatment or because of carious lesions. Periodontally diseased tissue was taken from teeth not worthy of preservation (n = 16) that had to be extracted because of chronic periodontitis. The generally accepted clinical parameters (probing depths, BOP, loss of attachment, tooth loosening and radiographic bone loss) were used for precise differentiation of the two samples. Each sample was also examined by real-time (RT) PCR with regard to the concentrations of nuclear factor kappa b (NF-?B). NF-?B is a marker of inflammation, which plays an important role in regulating the immune response in inflammation and is active in various inflammatory processes including perio-dontitis and periimplantitis [21]. The periimplant tissue samples consisted of tissue from 16 endosteal implants (Astra Tech, ITI Bonefit, Brånemark), which were explanted on account of advanced periimplantitis. In these cases, too, the findings described above were recorded for the patients, along with a detailed medical history. All participating patients were informed about the background to this study and gave written consent for use of the samples. The study was approved by the ethics committee of the medical faculty of Göttingen University.

Molecular biology methods of tissue preparation

Tissue from the lower third of the root surface and implant surface was obtained for tissue preparation, as PDL cells may still be present there especially in the case of teeth damaged by periodontal disease. This can be explained by the fact that bone atrophy has not yet occurred. The total RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany). The isolation was carried out according to the manufacturer’s RNeasy Mini instructions. To allow subsequent precise quantitative determination of gene expression, the samples were treated with DNase I (RNase-free) (Ambion, Austin, USA) [12].

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